As medical science continues to advance, the uses for isolated DNA and the desire for increased quantities of isolated DNA have led to a number of different methods for its isolation. Isolated DNA is employed in numerous applications, including gene discovery, disease diagnostics, drug discovery, the forensic sciences, and other research and clinical applications, including recombinant DNA research, cloning, sequencing, etc., using techniques such as hybridization, amplification, etc. Typically, DNA is isolated from cells in three sequential stages: (1) cells are lysed to release their content which includes protein, lipids, RNA and DNA; (2) ribonucleases (RNases) are optionally added to remove RNA; and (3) non-DNA contaminants such as protein are removed to yield pure DNA.
Methods for isolating nucleic acids from biological samples such as blood, cultured cells, tissues or body fluids typically are initiated by adding the biological sample directly to a detergent-containing lysis solution or a chaotropic-containing lysis solution sometimes also in the presence of a particulate solid phase (e.g., WO 96/18731 (Deggerdahl et al.), U.S. Pat. No. 5,234,809 (Boom et al.)). Alternatively, cells may be concentrated first by centrifugation and the suspended in a suspension solution. Achieving a uniform suspension of the compacted cells improves nucleic acid isolation by allowing more uniform contact with the detergent and/or chaotropic reagents that disrupt cell membranes and structures. Typical suspension solutions are hypotonic, such as that used in a pretreatment step to lysis red blood cells in mammalian blood (U.S. Pat. No. 5,777,098 (Gray), U.S. Pat. No. 5,973,137 (Heath et al.) or isotonic solutions such as phosphate buffered saline (e.g., WO 96/18731 (Deggerdahl et al.)), glucose (WO 97/10331 (Gonzales)) or sorbitol (U.S. Pat. No. 5,973,137 (Heath et al.)).
Several lysing reagents have been formulated to lyse cells. A lysate is created by mixing a biological sample comprising cells or viruses with the lysing reagent, by grinding tissue samples with a pestle in the presence of the lysing reagent thus facilitating penetration of the lysing reagent into the cells, or by dissociating tissue samples through mechanical or other means (for example, using sonication). The lysing reagent typically contains a detergent to disrupt cell membranes and solubilize proteins and lipids. The most common detergents used in lysing reagent formulations are the anionic detergents sodium dodecyl sulfate (SDS) and N-Lauroyl sarcosine as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., 7.3-7.24, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al. (Current Protocols in Molecular Biology, 4.0-4-4.5.3 and 13.12.1-13.12.3, John Wiley & Sons, New York (1989)). Non-ionic and cationic detergents have also been described for this purpose by Favaloro et al. (Methods Enzymol, 65, 718-749 (1980)) and U.S. Pat. No. 5,010,183 (MacFarlane) respectively.
After lysis, DNA is generally purified by separating it from the complex lysate, which contains non-DNA cellular material such as RNA, lipids and protein. The lysate is generally mixed with an organic solvent—typically, phenol and/or chloroform. Phenol not only denatures proteins but, following centrifugation, causes the protein to collect at the interface between the organic and aqueous layers; chloroform facilitates the separation of organic and aqueous phases. Such reagents, however, are typically unstable during storage due to oxidation. Moreover, these reagents are hazardous chemicals. Toxicity of such reagents is indicated by their LD50 values. The lower the LD50 value, the more hazardous the compound. Generally, lysing and/or purification solutions contain chloroform, which is highly toxic and a known carcinogen, having an LD50 of 908 mg/kg (rat oral administration). Phenol is highly toxic as well, having an LD50 of 317 mg/kg (mouse oral administration). A method for DNA isolation that uses less hazardous compounds, such as benzyl alcohol to replace phenol and chloroform, is disclosed by (U.S. Pat. No. 5,393,672 (Ness et al.)). However, despite the lower toxicity of benzyl alcohol, it is still classified as harmful with an LD50 of 1230 mg/kg by rat oral administration. Furthermore, even less toxic organic solvents require special handling and disposal.
Yet another major problem with conventional DNA isolation methods is the extensive labor and time required. The isolation of DNA from biological samples has been and continues to be labor intensive, requiring time consuming and repetitive tasks that occupy the bulk of a technician's time, often to the exclusion of other tasks. Currently, manual processes for the isolation of DNA require a time intensive operation of up to 24 hours. Excluding any incubation period, a technician may be required to perform upward of twenty tasks on a regular basis during the isolation process. The repetitive yet delicate process steps of DNA isolation require precision and attention to detail, and the relative success and/or yield may often rely on the skill of the technician responsible for the isolation. Repetitive application of precise process steps lends itself to errors which may negatively affect the quality and/or quantity of DNA isolated from a sample, or result in contamination of the sample. Furthermore, the generally large number of process steps required to isolate DNA increases the risk for contamination and cross-contamination of samples. In the case of unique or limited samples, such errors may occur when dealing with samples that cannot be duplicated, or are irreplaceable.
There exists a need for a method for the isolation of DNA from biological samples that does not employ harmful toxic reagents, and that employs fewer steps than conventional methods.